Expression of Nell-1 in vitro significantly increased osteoblast differentiation and mineralization. Overexpression of Nell-1 in transgenic mice significantly increased calvarial bone formation. Comparison of Nell-1 with BMP2 demonstrated comparable osteoinductive properties in a rodent calvarial defect model. In addition, Nell-1 exhibited a potential additive or synergistic effect with bone morphogenetic proteins (BMP2) on inducing alkaline phosphatase activity. Overall, we hypothesize that Nell-1 can enhance calvarial bone regeneration and perhaps enhance BMP mediated bone regeneration. To test this hypothesis, we have proposed two Aims. [unreadable] Aim 1 will separately optimize the concentrations of Nell-1, BMP2, and BMP7 required for bone formation (in a rat calvarial critical sized defect model) and then determine the effects of optimized Nell-1, BMP2, or BMP7 addition on specific cellular differentiation states (i.e., proliferation and apoptosis) and bone marker gene expression. In Aim 1A, our working hypothesis is Nell-1, BMP2, or BMP7 concentrations for optimized bone formation will vary. The optimal monotherapy dose (DoseOPT) for each factor will serve as an upper in vivo threshold for the in vivo combinations proposed in Aim 2B. In Aim 1B, our working hypothesis is that the cell type/differentiation stage can significantly impact the bioresponsiveness to optimized Nell-1 or BMPs. By corollary, Nell-1 and BMPs may induce bone through distinct pathways that involve differences in cell phenotype/differentiation state. To assess this, we will histologically co-localize osteoblastic differentiation marker expression with proliferative and apoptotic cellular phenotypes. [unreadable] Aim 2 will explore the effects of combinatorial Nell-1 and BMP formulations in vitro on specific parameters such as proliferation, osteoblast marker expression, apoptosis, and mineralization capacity and then attempt to translate these formulations in vivo using the same calvarial model as Aim 1. For Aim 2A, our working hypothesis is that specific Nell-1/BMP2 or Nell-1/BMP7 combination ratios in vitro will induce more mineralization than Nell-1, BMP2, or BMP7 alone. Optimized Nell-1/BMP combinations in Aim 2A will serve as a suggested Nell-1:BMP ratio for dosing in Aim 2B. For Aim 2B, our working hypothesis is that in vitro optimized Nell-1:BMP ratios can be combined with the in vivo optimized DoseOPT data from Aim 1 to better predict optimal in vivo Nell-1/BMP dose requirements for Aim 2B. The cellular and molecular analyses data from Aims 1B/2A2 will further fine tune Nell-1/BMP dosing in Aim 2B.Project Narrative [unreadable] [unreadable] [unreadable]